Reactive oxidizing species (ROS) are produced by many agents used in the treatment of cancer, e.g., ionizing radiation, photodynamic therapy and some chemotherapy drugs. ROS-mediated processes also play critical roles in tumor development by initiating and participating in signaling cascades leading to mitogenesis and tumor promotion, and by causing mutations. On the other hand, ROS are also vital to some normal physiological processes. The central question addressed by this Program Project Grant application is: How do reactive oxidizing species initiate such diverse cellular responses? Not all ROS are created equal. The cellular responses they initiate depend on ROS type (and hence reactivity), subcellular site of ROS generation, ROS concentration, and mode of signal transmission from the site of ROS generation to the site of response. In this revised Program, we will analyze the effects of ROS on cytotoxicity and cell signaling (Project 1 ), apoptosis (Project 2), and DNA damage induction and mutagenesis (Project 3). The type of ROS (1O2, .OH or H2O2) and its site of formation are expected to affect all these responses. Cellular signaling by diffusion of the initial ROS itself, secondary ROS, products of the ROS, or cascades of biomolecular changes initiated by ROS will be addressed. Core A will provide state-of-the-art facilities and expertise that are indispensable for all projects for subcellular production of ROS using laser light and photoactive agents, monitoring ROS, and cell imaging and analysis. The Gray Cancer Institute provides the ionizing radiation microbeams, capable of delivering spatially-resolved ionizing radiation to individual cells or subcellular regions. The proposed PPG has brought together a highly interactive, interdisciplinary team of experienced investigators using sophisticated tools to address questions that are fundamental to understanding molecular mechanisms of oxidative stress as it relates to cancer development and treatment. This is expected to result in increased insight that ultimately will fill an urgent need by translating into improved methods to prevent or treat cancer.